Co-Administration of Metformin and N-Acetyl Cysteine Fails to Improve Clinical Manifestations in PCOS Individual Undergoing ICSI.

BACKGROUND
Studies have demonstrated the efficacy of metformin (MTF ) in reducing insulin resistance and N-acetyl cysteine (NAC) in inhibiting oxidative stress which are involved in the pathogenesis of polycystic ovarian syndrome (PCOS). We aimed to compare the effects of MTF and NAC combination on serum metabolite and hormonal levels during the course of ovulation induction in PCOS individual candidates of intracytoplasmic sperm injection (ICSI).


MATERIALS AND METHODS
In this prospective randomized clinical trial, placebo con- trolled pilot study, 80 patients of polycystic ovarian syndrome at the age of 25-35 years were divided into 4 groups (n=20): i. NAC=treated with N-acetyl cysteine (600 mg three times daily), ii. MTF=treated with metformin (500 mg three times daily), iii. MTF+NAC=treated with N-acetyl cysteine plus metformin (the offered doses) and iv. placebo (PLA). A total number of 20 patients (6 from MTF group, 4 from NAC group, 6 from MTF+NAC group and 4 from PLA group) were dropped of the study. The drugs were administrated from day 3 of menses of previous cycle until ovum pick-up.


RESULTS
Serum levels of luteinizing hormone (LH), total testosterone, cholester- ol and triglyceride, insulin and leptin significantly reduced in the MTF and NAC groups compared to the placebo (p<0.01). But levels of LH, total testosterone, cholesterol and triglyceride had no significant reduction in the MTF+NAC groups compared to the placebo. The serum levels of malonyldialdehyde (MDA), insulin and leptin reduced significantly after treatment in the MTF+NAC group compared to the placebo (p<0.05).


CONCLUSION
CONSIDERING THE ADVERSE EFFECT OF COMBINATION THERAPY, WE PROPOSED THE CONADMINISTRATION MIGHT HAVE NO BENEFICIAL EFFECT FOR PCOS PATIENT DURING COURSE OF OVULATION INDUCTION OF ICSI (REGISTRATION NUMBER: IRCT201204159476N1).


Introduction
Polycystic ovarian syndrome (PCOS) is an ovarian dysfunction syndrome with the combination of heterogeneous symptoms and signs; it is considered the most prevalent endocrinopathy resulting in anovulation up to 10% of women at reproductive age (1). A rise in the serum levels of androgen, insulin and luteinizing hormone (LH), menstrual dysfunction, hirsutism, infertility and obesity are some of the complications which are associated with PCOS (2). In addition, endometrial hyperplasia, cancer, type II diabetes, hypertension and dyslipidemia are considered as long term consequences of this syndrome (3). Studies have concluded that treatment with insulin-sensitizing agents such as metformin (MTF) results in a decrease of the levels of serum lipids, androgen and insulin; regulation of menstrual cycles; and promotion of both spontaneous and induced ovulation which leads to an increase in pregnancy rate (4). MTF has been used in the treatment of PCOS for a long time; however, gastrointestinal side effects, hypoglycemia and an increase in the serum homocysteine levels in some patients are associated with consuming MTF (5, 6). Hyperhomocysteinaemia is regarded as risk factors for cardiovascular diseases, thrombophilia, pre-eclampsia and recurrent abortion (7,8).
N-acetyl cysteine (NAC) is an acetylated variant of L-cysteine containing sulfhydryl groups which acts as a powerful antioxidant, antiapoptotic and free-radical scavenger; it also stimulates production of glutathione (9, 10), lowers the serum homocysteine levels and promotes detoxification (11). Since clinical evidence suggests that hyperhomocysteinaemia leads to endothelial dysfunction through generation of reactive oxygen species (12), NAC can preserve vascular integrity and protect against ischemic injuries (13).
Since the effects of co-administration of MTF and NAC compared to MTF and NAC alone in reducing metabolic and hormonal tients with PCOS has not been evaluated yet, we have evaluated and compared the efficacy of co-treatment of MTF and NAC on clinical, metabolic and hormonal aspects during course of ovulation induction in PCOS individual undergoing ICSI cycle.

Study population
Through a prospective randomized clinical trial, placebo controlled pilot study, in the interval between July 2012 and February 2013, 80 infertile PCOS individuals at the age of 25-35 years, candidate of ICSI, were enrolled. This study was conducted in the in vitro fertilization (IVF) Unit of Infertility Research Center of the ACECR, Qom.
Patients needed to fulfill PCOS diagnostic criteria which was based on the Rotterdam having two out of three criteria of chronic oligo-or anovulation, clinical or biochemical hyperandrogenism, or polycystic ovaries at sonography examination. Hypersensitivity to either MTF or NAC, presence of infertility factors other than anovulation, male infertility, pelvic organic pathologies, congenital adrenal hyperplasia, thyroid dysfunction, Cushing's syndrome, hyperprolactinemia, androgen secreting neoplasia, diabetes mellitus, consumption of medications affecting carbohydrate metabolism, taking hormonal analogues other than progesterone two months prior to enrollment, and severe hepatic or kidney diseases were considered as exclusion criteria. The semen samples were assessed according to WHO guidelines (15) and individuals with abnormal semen parameters were excluded from study.
An approval from the Research Ethics Committee of Royan Institute, Iran, and an informed consent from participants were obtained. All patients were asked to avoid any changes in their normal physical activity and diet and also not to take any new pharmacotherapy during the study.

Treatment design
The patients were examined by the gynecologists. The subjects were randomly selected to receive either MTF and NAC or placebo. Eighty patients were divided into four received NAC (Holzkrichen, Germany, batch no. 6N5483; 600 mg three times daily), ii. MTF group which received MTF (Glucophage, daily), iii. MTF+NAC group which were cotreated with MTF and NAC with the offered doses three times daily, and iv. placebo (PLA group) which received oral rehydration salts (ORS, Poursina, Tehran, Iran; batch no.30) three times daily, for a period of six weeks. Before beginning treatment, patients were also asked to report any possible adverse effects during the treatment, and they were evaluated for any presenting main complaints at the end of the treatment period.
A total number of 20 patients (6 from MTF group, 4 from NAC group, 6 from MTF+NAC group and 4 from PLA group) were dropped out due to intolerance of medication, monofollicular development, and failure of ovulation induction during ICSI cycle, and ultimately, 60 patients remained in the study.

Ovulation induction
Patients with PCOS undergoing ICSI treatment using a long GnRH agonist protocol received MTF, NAC, or MTF+NAC tablets, randomly, from third day of menses of previous cycle until the day of oocyte aspiration.
All patients received oral contraceptive pills (OCPs) for 21 days starting simultaneously with MTF, NAC or MTF+NAC on day 3 of menses of the cycle prior to the treatment cycle. Ovarian down-regulation was initiated with daily buserelin acetate 1 mg (Suprefact, Aventis, Germany), beginning on day 19 of the preceding menstruation (Luteal phase) and after ovarian down-regulation was achieved day 2 of last menstrual period (LMP) and the dose was then reduced to 0.5 mg when the thickness of the endometrium was <4 mm. Ovarian stimulation began with daily injections of average 2 ampoule of recombinant follicle stimulating hormone (rFSH; Gonal-f, Merck Serono S.A., Geneva, Switzerland). Following cycle monitoring, using vaginal sonography (HS 4000, Honda Electronics Co., Japan), ovulation was induced by the administration of average 10,000 IU human chorionic gonadotropin (HCG; Pregnyl, Organon, Netherlands). When at least three follicles had reached diameters of 16-18 mm, transvaginal oocyte aspiration was performed with ultrasound guidance under general anesthesia 36 hours after injection of HCG.

Assessment of baseline and clinical features
The body mass index (BMI), the waist/hip ratio (WHR) and the blood pressures were recorded for each patient once before the treatment on the third day of menstruation and once in the day of ovum pick up (OPU) of ICSI cycle. Fasting blood samples were collected from each individual once before the treatment on day 3 of menses of previous cycle before ICSI and once during oocyte aspiration. The peripheral blood sample taken from each patient were immediately centrifuged for 10 minutes at 3000 rpm (EBA20, Hettich, UK), and serum tion and analysis.

Statistical analysis
The normality of continuous variables were were analyzed with one-way ANOVA and the Tukey's test for post-hoc analysis. Chi-squared test was used for the statistical analysis where ly different at p<0.05. Pearson's correlation test variables. Multivariate linear regression analysis was done to test the effect of leptin on hyperinsulinemia and on increased stress oxidative. All data were analyzed with a Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) version 16.

Clinical and demographic characteristics
duration of marriage, duration of infertility, the occurrence of oligomenorrhoea, amenorrhoea and hirsutism between the four groups are presented in table 1.
weight, height, BMI, waist size, hip size and WHR in the treatment groups are presented in table 2.

Biochemical characteristics
Serum levels of LH, total testosterone, cholesterol and triglyceride, insulin and leptin significantly reduced in the MTF and NAC groups compared to the placebo (p<0.05). But levels of LH, total testosterone, cholesterol and triglyceride had no significant reduction in the MTF+NAC groups compared to the placebo (p>0.05). The serum levels of insulin and lep-tin reduced significantly after treatment in the MTF+NAC group compared to the placebo (p<0.01). Unlike the MTF+NAC and NAC not significantly decreased compared to placebo group (p>0.05). Levels of FBS, FSH, PRL, tio were not significantly different in treatment groups and to those of the placebo group, data are presented in table 3.

for the comparison between treatments are in bold type. a; Differences were observed between in the experimental groups compared with placebo, b; MTF group compared with placebo 6 weeks after treatment (p<0.05), c; NAC group compared with placebo 6 weeks after treatment (p<0.05), d; MTF+NAC group compared with placebo 6 weeks after treatment (p<0.05) NS; No differences were observed between the mean values of variables in the experimental groups compared
Upon performing multivariate analyses after adrelationship between leptin and both insulin (p<0.01) Considering the amount of data and length of the manuscript, the data regarding clinical outcomes of ICSI will be presented in a different manuscript.

Discussion
PCOS has been a research subject over the past six decades, while it is associated with insulin resistance, hyperandrogenism, obesity, dyslipidemia an insulin sensitizer (5-7), and NAC (9, 11, 17) on biochemical and clinical aspects of PCOS have been demonstrated by many studies. MTF appears to reduce the output of hepatic glucose, declining insulin concentrations and reducing the androgen production by theca cells (18). On the other hand, NAC is commonly used as a safe drug which increases the cellular levels of reduced glutathione. index values (9, 17).
sumption in patients with PCOS, we decided to investigate whether a combination therapy of MTF and NAC during the course of ovarian stimulation during ICSI cycle in patients with PCOS will introduce a better effect in reducing hyperinsulinism and hyperandrogenism and in normalizing other biochemical and endocrine parameters rather than using MTF or NAC alone. We should stress that our study is a pilot study, and there is a lack of literature on combined administration of these drug, especially during course of ovarian stimulation.
Our results showed that 6 weeks of co-treatment reducing the levels of insulin compared with the control. Although this result supports previous in the fasting insulin levels after the consumption of MTF and NAC compared with the placebo level of insulin following combination therapy. There is also some contrary reports (9,19,20) which could probably be due to the differences in patient inclusion criteria, dosing and duration of therapy, and genetic background of individuals.
Concomitant with reduced hyperinsulinaemia, upon treatment with MTF, NAC and MTF+NAC groups was observed compared to the control group. This observation was consistent with background literature on MTF and NAC (21). These observations are in line with previous report on leptin which is considered to play an important role in production (22). Furthermore, in our study, a sigleptin and insulin concentrations before and after treatment, indicating that insulin reduction was responsible for lowering leptin secretion. Moreover, it should be emphasized that when multiple regression analysis was performed, such correlation was found. Other study (23) have also shown and positively with insulin in women with PCOS. On the other hand, in a study of Yarali et al. (19), six weeks of treatment with 850 mg MTF twice levels compared to the placebo, which is probably due to the differences in drug doses and protocols of ovulation induction.
the levels of TT after 6 weeks of treatment with NAC and MTF alone, which is in consistent with androgen production from theca-cells by increasing the activity of cytochrome P450-C17a, and it also induces adrenal esteroidogenesis (16). Therefore, MTF lowers the level of serum androgens through direct inhibition of androgen production in human ovarian theca cells and also by improving insulin sensitivity, indirectly (24). On the other hand, NAC decreases the expression of several key enzymes involved in T production such as and leading to a decrease in the levels of T production. However, in contrary to our expectations, MTF decrease in testosterone levels, probably due to the effects of drug interference which need future investigation. In our study, as well as previous study and positively with TT in PCOS patients, which likely suggests that a decrease in leptin levels can reduce esteroidogenesis. An abnormal lipid prosimilar to previous study (27), an improved lipid administration by means of reduced TG and total cholesterol. These effects were not observed in combination therapy, and we have no suggestion for these observations, but drug co-treatment re-mains to be explored and needs future validation.
Several studies have pointed out an increase in oxidative stress in PCOS patients which is in-mocysteinemia, hyperandrogenemia and insulin resistance (28,29). Leptin has peripheral actions stress and cardiovascular disease (30). Leptin via Janus kinases(JNK) pathway [one of the intracellular signaling pathways; mitogen-activated protein activity of NF-kB (nuclear factor-kB) and AP-1(activator protein-1), thereby increases reactive oxygen species (ROS) through induction of cellu--er of lipid-peroxidation and of increased oxidative stress, and the total testosterone and insulin levels in PCOS patients was observed in this study, conin patients with PCOS (32). In our study, follow-pared to MTF group. This could be due to the fact that MTF may raise oxidative stress through increased homocysteine levels which is associated with increased generation of superoxide anions and decreased activity of antioxidant enzymes (32). Since NAC, as an antioxidant, inhibits the transcription factors activity of NF-kB and AP-1, of MAP-kinase and results in a reduction in the oxidative stress (11-13); therefore, it is possible that NAC can decrease elevated oxidative stress in PCOS patients taking MTF.
after 6 weeks of treatment with NAC and MTF ings (19) about MTF+NAC group as compared with the control. However, the reduction of AMH level in the treatment groups, which was close erature, so it has been suggested that long-term administration is required for a reduction in the raised AMH level observed in PCOS individuals (33), indicating that these drugs can reduced the number of AMH producing follicle which is appropriate for PCOS patient.
Conclusion different influence of action (i.e. one as insulin sensitizers and other as an antioxidant), we observed no beneficial effect of these drugs which administered together during course of ovulation induction for ICSI. In contrast to our expectation, the beneficial effect of each drug on reducing LH, TT, cholesterol and TG which was observed when MTF or NAC was administered alone rather than combination therapy. Therefore, we conclude that co-administration of these drugs might not be useful for PCOS patient during course of ovulation stimulation. However, the beneficial effects of co-administration of the two drugs for routine treatment of PCOS patient remain to be evaluated.